Glucagon-like insulinotropic peptides, compositions and methods

ABSTRACT

The present invention provides novel complexes consisting of certain GLP-1 molecules associated with a divalent metal cation that is capable of co-precipitating with a GLP-1 molecule. Pharmaceutical compositions and methods of using such complexes for enhancing the expression of insulin in B-type islet cells is claimed, as is a method for treating maturity onset diabetes mellitus in mammals, particularly humans.

This application is a division of application Ser. No. 08/407,831, filedMar. 21, 1995, now U.S. Pat. No. 5,705,483, which is acontinuation-in-part of Galloway et al., U.S. Ser. No. 08/164,277, filedDec. 9, 1993, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the field of pharmaceutical and organicchemistry and provides novel compounds, and pharmaceutical compositionsthereof, which are useful for enhancing the expression of insulin frommammalian pancreatic B-type islet cells and for treating maturity onsetdiabetes mellitus in a mammal.

The endocrine secretions of the pancreatic islets are under complexcontrol not only by blood-borne metabolites (glucose, amino acids,catecholamines, etc.), but also by local paracrine influences. The majorpancreatic islet hormones (glucagon, insulin and somatostatin) interactamongst their specific cell types (A, B, and D cells, respectively) tomodulate secretory responses mediated by the aforementioned metabolites.Although insulin secretion is predominantly controlled by blood levelsof glucose, somatostatin inhibits glucose-mediated insulin secretoryresponses. In addition to the proposed interislet paracrine regulationof insulin secretion, there is evidence to support the existence ofinsulinotropic factors in the intestine. This concept originates fromthe observations that glucose taken orally is a much more potentstimulant of insulin secretion than is a comparable amount of glucosegiven intravenously.

The human hormone glucagon is a 29-amino acid peptide hormone producedin the A-cells of the pancreas. The hormone belongs to a multi-genefamily of structurally related peptides that include secretin, gastricinhibitory peptide, vasoactive intestinal peptide and glicentin. Thesepeptides variously regulate carbohydrate metabolism, gastrointestinalmobility and secretory processing. The principal recognized actions ofpancreatic glucagon, however, are to promote hepatic glycogenolysis andglyconeogenesis, resulting in an elevation of blood sugar levels. Inthis regard, the actions of glucagon are counter regulatory to those ofinsulin and may contribute to the hyperglycemia that accompaniesDiabetes mellitus [(Lund, P. K., et al., Proc. Natl. Acad. Sci. U.S.A.,79:345-349 (1982)].

Glucagon has been found to be capable of binding to specific receptorswhich lie on the surface of insulin producing cells. Glucagon, whenbound to these receptors, stimulates the rapid synthesis of cAMP bythese cells. cAMP, in turn, has been found to stimulate insulinexpression [Korman, L. Y., et al., Diabetes, 34:717-722 (1985)]. Insulinacts to inhibit glucagon synthesis [Ganong, W. F., Review of MedicalPhysiology, Lange Publications, Los Altos, Calif., p. 273 (1979)]. Thus,the expression of glucagon is carefully regulated by insulin, andultimately by the serum glucose level.

The glucagon gene is initially translated from a 360 base pair precursorto form the polypeptide, preproglucagon [Lund, et al., Proc. Natl. Acad.Sci. U.S.A. 79:345-349 (1982)]. This polypeptide is subsequentlyprocessed to form proglucagon. Patzelt, C., et al., Nature, 282:260-266(1979), demonstrated that proglucagon was subsequently cleaved intoglucagon and a second polypeptide. Subsequent work by Lund, P. K., etal., Lopez L. C., et al., Proc. Natl. Acad. Sci. U.S.A., 80:5485-5489(1983), and Bell, G. I., et al., Nature 302:716-718 (1983), demonstratedthat the proglucagon molecule was cleaved immediately afterlysine-arginine dipeptide residues. Studies of proglucagon produced bychannel catfish (Ictalurus punctata) indicated that glucagon from thisanimal was also proteolytically cleaved after adjacent lysine-argininedipeptide residues [Andrews P. C., et al., J. Biol. Chem., 260:3910-3914(1985), Lopez, L. C., et al., Proc. Natl. Acad. Sci. U.S.A.,80:5485-5489 (1983)]. Bell, G. I., et al., supra, discovered thatmammalian proglucagon was cleaved at lysine-arginine orarginine-arginine dipeptides, and demonstrated that the proglucagonmolecule contained three discrete and highly homologous peptidemolecules which were designated glucagon, glucagon-like peptide 1(GLP-1) and glucagon-like peptide 2 (GLP-2). Lopez, et al., concludedthat glucagon-like peptide 1 was 37 amino acid residues long and thatglucagon-like peptide 2 was 34 amino acid residues long. Analogousstudies on the structure of rat preproglucagon revealed a similarpattern of proteolytic cleavage between adjacent lysine-arginine orarginine-arginine dipeptide residues, resulting in the formation ofglucagon, GLP-1 and GLP-2 [Heinrich, G., et al., Endocrinol.,115:2176-2181 (1984)]. Human, rat, bovine, and hamster sequences ofGLP-1 have been found to be identical [Ghiglione, M., et al.,Diabetologia, 27:599-600 (1984)].

The conclusion reached by Lopez, et al., regarding the size of GLP-1 wasconfirmed by the work of Uttenthal, L. O., et al., J. Clin. Endocrinol.Metabol., 61:472-479 (1985). Uttenthal, et al., examined the molecularforms of GLP-1 which were present in the human pancreas. Their researchshows that GLP-1 and GLP-2 are present in the pancreas as 37 amino acidand 34 amino acid peptides, respectively.

The similarity between GLP-1 and glucagon suggested to earlyinvestigators that GLP-1 might have biological activity. Although someinvestigators found that GLP-1 could induce rat brain cells tosynthesize cAMP [Hoosein, N. M., et al., Febs Lett. 178:83-86 (1984)],other investigators failed to identify any physiological role for GLP-1(Lopez, L. C., et al.). The failure to identify any physiological rolefor GLP-1 caused some investigators to question whether GLP-1 was infact a hormone and whether the relatedness between glucagon and GLP-1might be artifactual.

Variants of GLP-1 (7-37) and analogs thereof, also have been disclosed.These variants and analogs include, for example, Gln⁹ -GLP-1 (7-37),D-Gln⁹ -GLP-1 (7-37), acetyl-Lys⁹ -GLP-1 (7-37), Thr¹⁶ -Lys¹⁸ -GLP-1(7-37), Lys¹⁸ -GLP-1 (7-37) and the like, and derivatives thereofincluding, for example, acid addition salts, carboxylate salts, loweralkyl esters, and amides [see, e.g., WO 91/11457]. Generally, thevarious disclosed forms of GLP-1 are known to stimulate insulinsecretion (insulinotropic action) and cAMP formation [see, e g., Mojsov,S., Int. J. Peptide Protein Research, 40:333-343 (1992)].

More importantly, multiple authors have demonstrated the nexus betweenlaboratory experimentation and mammalian, particularly human,insulinotropic responses to exogenous administration of GLP-1,particularly GLP-1 (7-36)NH₂ and GLP-1 (7-37) [see, e.g., Nauck, M. A.,et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., NewEngland J. of Medicine, 326(20):1316-1322 (1992); Nauck, M. A., et al.,J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes,42:1219-1225 (1993)].

More particularly, the fundamental defects identified as causinghyperglycemia in maturity onset diabetes are impaired secretion ofendogenous insulin and resistance to the effects of insulin by muscleand liver [Galloway, J. S., Diabetes Care, 13:1209-1239, (1990)]. Thelatter defect results in excessive production of glucose from the liver.Thus, whereas a normal individual releases glucose at the rate ofapproximately 2 mg/kg/minute, in patients with maturity onset diabetes,this amount usually exceeds 2.5 mg/kg/minute resulting in a net excessof at least 70 grams of glucose per 24 hours. The fact that there existsexceedingly high correlations between hepatic glucose production, thefasting blood glucose and overall metabolic control as indicated byglycohemoglobin measurements [Galloway, J. A., supra; and Galloway, J.A., et al., Clin. Therap., 12:460-472 (1990)], it is readily apparentthat control of the fasting blood glucose is a sine quo non forachieving overall normalization of metabolism sufficient to prevent thecomplication of hyperglycemia. In view of the fact that present forms ofinsulin rarely normalize hepatic glucose production without producingsignificant hyperinsulinemia and hypoglycemia (Galloway, J. A., andGalloway, J. A., et al., supra) alternative approaches are needed.

Intravenous infusions of GLIP-1 (7-36)NH₂ to produce twice normal serumconcentrations have been demonstrated to produce the effects indicatedin the table below:

    ______________________________________                                                      Normal  Patients With Maturity                                                         Subjects Onset Diabetes                                ______________________________________                                        Meal glycemia (1)                                                                             Unchanged Reduced                                               Fasting glycemia (2) --  Reduced                                              Fasting glucagon (2) -- Reduced                                               Post-prandial glucagon (1) -- Reduced                                         Endogenous insulin secretion Unchanged Increased                              in response to a meal (1)                                                     Free fatty acids Reduced (3) Reduced (2)                                    ______________________________________                                         (1) Gutniak, M., et al., supra.                                               (2) Nauck, M. A., et. al., Diabetologia, supra.                               (3) Orskov, C., et al., Diabetes, 42:658-661, (1993).                    

However, the long-term stability of GLP-1, particularly GLP-1 as acomponent of a pharmaceutical composition for administration to mammals,is questionable. In fact, when stored at the low temperature of 4° C.,by-products of GLP-1 (7-37) have been found as early as eleven monthsafter sample preparation (Mojsov, S., supra). Thus, there exists a needfor a more stable GLP-1 compound which can safely be administered tomammals in need of such treatment.

Furthermore, the biological half-life of GLP-1 molecules, particularlythose molecules which are affected by the activity ofdipeptidyl-peptidase IV (DPP IV) is quite short. For example, thebiological half-life of GLP-1 (7-37) is a mere 3 to 5 minutes (U.S. Pat.No. 5,118,666), and is further influenced by its rapid absorptionfollowing parenteral administration to a mammal. Thus, there also existsa need for a GLP-1 compound which delays absorption following suchadministration.

Accordingly, the present invention provides compounds which satisfy theaforementioned stability requirements. The compounds of the presentinvention also provide delayed absorption following parenteraladministration and, consequently, should have extended biologicalhalf-lives. Also provided are pharmaceutical compositions of thecompounds of the present invention, as well as methods for using suchcompounds.

SUMMARY OF THE INVENTION

The present invention provides a complex consisting of a divalent metalcation associated with and capable of co-precipitating with a compoundof the formula:

    R.sub.1 -X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sub.2

wherein:

R₁ is selected from the group consisting of L-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;

X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, andalpha-methyl-Ala;

Y is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, andGly;

Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, andGly;

R₂ is selected from the group consisting of NH₂, and Gly-OH; providingthat the compound has an isoelectric point in the range from about 6.0to about 9.0 and further providing that when R₁ is His, X is Ala, Y isGlu, and Z is Glu, R₂ must be NH₂.

Also provided by the present invention is a pharmaceutical compositioncomprising a compound of the present invention in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.

The present invention further provides a method for enhancing theexpression of insulin comprising providing to a mammalian pancreaticB-type islet cell an effective amount of a compound of the presentinvention, as well as a method of treating maturity onset diabetesmellitus which comprises administering to a mammal in need of suchtreatment an effective amount of a compound of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a complex consisting of aGLP-1 molecule having an isoelectric point in the range from about 6.0to about 9.0, complexed with a divalent metal cation.

As used in the present specification, the term "GLP-1 molecule" refersto naturally-occurring GLP-1 (7-36)NH₂, GLP-1 (7-37), natural andunnatural functional analogs and derivatives thereof, and salts thereof.The amino acid sequence of GLP-1 (7-36)NH₂ is well known in the art, butis presented below as a convenience to the reader:

    His.sup.7 -Ala-Glu-Gly.sup.10 -Thr-Phe-Thr-Ser-Asp.sup.15 -Val-Ser-Ser-Tyr-Leu.sup.20 -Glu-Gly-Gln-Ala-Ala.sup.25 -Lys-Glu-Phd-Ile-Ala.sup.30 -Trp-Leu-Val-Lyp-Gly.sup.35 -Arg-NH.sub.2. (SEQ ID NO:1).

For GLP-1 (7-37), the carboxy-terminal amide functionality of Arg³⁶ isdisplaced with Gly at the 37th position of the GLP-1 (7-36)NH₂ molecule.

In addition, the existence and preparation of a multitude of protected,unprotected, and partially protected natural and unnatural functionalanalogs and derivatives of GLP-1 (7-36)NH₂ and GLP-1 (7-37) moleculeshave been described in the art [see, e.g., U.S. Pat. No. 5,120,712 andU.S. Pat. No. 5,118,666, which are herein incorporated by reference, andOrskov, C., et al., J. Biol. Chem., 264(22):12826-12829 (1989) and WO91/11457 (Buckley, D. I., et al., published Aug. 8, 1991)].

As known in the art, amino acid residues may be in their protected formin which both amino and carboxy groups possess appropriate protectinggroups, partially-protected form in which either amino or carboxy groupspossess appropriate protecting groups, or unprotected form in whichneither amino nor carboxy groups possess an appropriate protectinggroup. Numerous reactions for the formation and removal of suchprotecting groups are described in a number of standard works including,for example, "Protective Groups in Organic Chemistry", Plenum Press(London and New York, 1973); Green, T. H., "Protective Groups in OrganicSynthesis", Wiley (New York, 1981); and "The Peptides", Vol. I, Schroderand Lubke, Academic Press (London and New York, 1965).

Representative amino protecting groups include, for example, formyl,acetyl, isopropyl, butoxycarbonyl, fluorenylmethoxycarbonyl,carbobenzyloxy, and the like.

Representative carboxy protecting groups include, for example, benzylester, methyl ester, ethyl ester, t-butyl ester, p-nitro phenyl ester,and the like.

In addition to protected forms in which both amino and carboxy groupspossess appropriate protecting groups, the term "protected" also refersto those GLP-1 molecules in which the activity of dipeptidyl-peptidaseIV is resisted or inhibited [see, e.g., Mentlein, R., et al., Eur. J.Biochem., 214:829-835 (1993)]. In addition to GLP-1(7-36)NH₂, moleculeswhich are protected from the activity of DPP IV are preferred, and Gly⁸-GLP-1(7-36)NH₂, Val⁸ -GLP-1(7-37)OH, α-methly-Ala⁸ -GLP-1(7-36)NH₂, andGly⁸ -Gln²¹ -GLP-1(7-37)OH are more preferred.

Derivatives of naturally-occurring GLP-1 molecules are those peptideswhich are obtained by fragmenting a naturally-occurring sequence, or aresynthesized based upon a knowledge of the sequence of thenaturally-occurring amino acid sequence of the genetic material (DNA orRNA) which encodes this sequence. The term "derivatives" also includeschemical modification of natural or unnatural GLP-1 molecules. Processesfor preparing these derivatives are well known to organic and peptidechemists of ordinary skill (see, e.g., WO 91/11457, supra).

GLP-1 molecules of the present invention also include analogs of GLP-1(7-36)NH₂ and GLP-1 (7-37) in which one or more amino acids which arenot present in the original sequence are added or deleted, andderivatives thereof. Specifically, His and desamino-histidine arepreferred for R₁, so long as the overall isoelectric point of themolecule is in the range of about 6 to 9. Ala, Gly, and Val arepreferred at the "X" position, so long as the overall isoelectric pointof the molecule is in the range of about 6 to 9. Likewise, Glu, and Glnare preferred at the "Y" position, so long as the overall isoelectricpoint of the molecule is in the range of about 6 to 9. Also, Glu, andGln are preferred at the "Z" position, so long as the overallisoelectric point of the molecule is in the range of about 6 to 9.Finally, Gly-OH is preferred for R₂, so long as the overall isoelectricpoint of the molecule is in the range of about 6 to 9.

Furthermore, the present invention includes a salt form of a GLP-1molecule. A GLP-1 of this invention can possess a sufficiently acidic, asufficiently basic, or both functional groups, and accordingly reactwith any of a number of inorganic bases, and inorganic and organicacids, to form a salt. Acids commonly employed to form acid additionsalts are inorganic acids such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, phosphoric acid, and the like, andorganic acids such as p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid, and the like. Examples of suchsalts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like. Preferred acid addition salts are those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, and,especially, hydrochloric acid.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like. The salt formsare particularly preferred.

Of course, when the compounds of this invention are used forpharmacotherapeutic purposes, those compounds may also be in the form ofa salt, but the salt must be pharmaceutically acceptable.

Thus, GLP-1 molecules of the present invention include inter alia, thoseGLP-1 molecules which functionally demonstrate insulinotropic activity.The term "insulinotropic activity" relates to the ability of a substanceto stimulate, or cause the stimulation of, the synthesis or expressionof the hormone insulin.

The insulinotropic property of a compound may be determined by providingthat compound to animal cells, or injecting that compound into animalsand monitoring the release of immunoreactive insulin (IRI) into themedia or circulatory system of the animal, respectively. The presence ofIRI is detected through the use of a radioimmunoassay which canspecifically detect insulin.

Although any radioimmunoassay capable of detecting the presence of IRImay be employed, it is preferable to use a modification of the assaymethod of Albano, J. D. M., et al., Acta Endocrinol., 70:487-509 (1972).In this modification, a phosphate/albumin buffer with a pH of 7.4 isemployed. The incubation is prepared with the consecutive addition of500 μl of phosphate buffer, 50 μl of perfusate sample or rat insulinstandard in perfusate, 100 μl of anti-insulin antiserum (WellcomeLaboratories; 1:40,000 dilution), and 100 μl of [¹²⁵) insulin, giving atotal volume of 750 μl in a 10×75 mm disposable glass tube. Afterincubation for 2-3 days at 4° C., free insulin is separated fromantibody-bound insulin by charcoal separation. The assay sensitivity is1-2 uU/mL. In order to measure the release of IRI into the cell culturemedium of cells grown in tissue culture, one preferably incorporatesradioactive label into proinsulin. Although any radioactive labelcapable of labelling a polypeptide can be used, it is preferable to use³ H leucine in order to obtain labelled proinsulin. Labelling can bedone for any period of time sufficient to permit the formation of adetectably labelled pool of proinsulin molecules; however, it ispreferable to incubate cells in the presence of radioactive label for a60 minute time period.

Although many cell lines capable of expressing insulin can be used fordetermining whether a compound has an insulinotropic effect, it ispreferable to use rat insulinoma cells, and especially RIN-38 ratinsulinoma cells. Such cells can be grown in any suitable medium;however, it is preferable to use DME medium containing 0.1% BSA and 25mM glucose.

The insulinotropic property of a compound may also be determined bypancreatic infusion. The in situ isolated perfused rat pancreaspreparation is a modification of the method of Penhos, J. C., et al.,Diabetes, 18:733-738 (1969). Fasted male Charles River strain albinorats, weighing 350-600 g, are anesthetized with an intraperitonealinjection of Amytal Sodium (Eli Lilly and Co.: 160 ng/kg). Renal,adrenal, gastric, and lower colonic blood vessels are ligated. Theentire intestine is resected except for about four cm of duodenum andthe descending colon and rectum. Therefore, only a small part of theintestine is perfused, minimizing possible interference by entericsubstances with glucagon-like immunoreactivity. The perfusate is amodified Krebs-Ringer bicarbonate buffer with 4% dextran T70 and 0.2%bovine serum albumin (fraction V), and is bubbled with 95% O₂ and 5%CO₂. A nonpulsatile flow, 4-channel roller bearing pump (Buchlerpolystatic, Buchler Instruments Division, Nuclear-Chicago Corp.) isused, and a switch from one perfusate source to another is accomplishedby switching a 3-way stopcock. The manner in which perfusion isperformed, monitored, and analyzed follow the method of Weir, G. C., etal., J. Clin. Inestigat. 54:1403-1412 (1974), which is herebyincorporated by reference.

The GLP-1 molecules of the present invention are required to possess ahistidine functionality at the amino terminus. GLP-1 molecules of thepresent invention may also possess a modified histidine functionality inlieu of the required histidine functionality.

The term "modified histidine" refers to a histidine functionality whichhas been chemically or biologically altered or an altered histidinefunctionality which has been synthesized de novo, but which retains itsmetal binding properties.

Numerous such modified histidine functionalities and their preparationare known in the art and include, for example, D-histidine (WO91/11457), desamino-histidine (WO 92/18531), 2-amino-histidine[Levine-Pinto, H., et al., Biochem. Biophys. Res. Commun.,103(4):1121-1130 (1981)], β-hydroxy-L-histidine [Owa, T, et al.,Chemistry Letters, pp. 1873-1874 (1988)], L-homohistidine [Altman, J.,et al., Synthetic Commun., 19(11&12):2069-2076 (1989)],α-fluoromethyl-histidine (U.S. Pat. No. 4,347,374), andα-methylhistidine [O'Donnell, M. J., Synthetic Commun., 9(7&8):1157-1165(1989)].

The GLP-1 molecules of the present invention further are required tohave an isoelectric point in the range from about 6.0 to about 9.0.Numerous GLP-1 molecules having an isoelectric point in this range havebeen disclosed and include, for example:

GLP-1 (7-36)NH₂

Gly⁸ -GLP-1 (7-36)NH₂

Gln⁹ -GLP-1 (7-37)

D-Gln⁹ -GLP-1 (7-37)

acetyl-Lys⁹ -GLP-1 (7-37)

Thr⁹ -GLP-1 (7-37)

D-Thr⁹ -GLP-1 (7-37)

Asn⁹ -GLP-1 (7-37)

D-Asn⁹ -GLP-1 (7-37)

Ser²² -Arg²³ -Arg²⁴ -Gln²⁶ -GLP-1 (7-37)

Thr¹⁶ -Lys¹⁸ -GLP-1 (7-37)

Lys¹⁸ -GLP-1 (7-37)

Arg²³ -GLP-1 (7-37)

Arg²⁴ -GLP-1 (7-37), and the like (see, e.g., WO 91/11457, supra). Inaddition, GLP-1 molecules of the present invention, when possessing eachof the above-referenced modified histidine functionalities in lieu ofthe histidine functionality, have isoelectric points which fall withinthe above-defined range. Methods for calculating or experimentallydetermining the isoelectric point of other GLP-1 molecules are known toone of ordinary skill in the art.

Methods for preparing the GLP-1 molecules of the present invention alsoare well known to an ordinarily skilled peptide chemist.

In one method, GLP-1 molecules are prepared by the well-known solidphase peptide synthetic schemes described by Merrifield, J. M., Chem.Soc., 85:2149 (1962), and Stewart and Young, Solid Phase PeptideSynthesis, pp. 24-66, Freeman (San Francisco, 1969). However, it also ispossible to obtain fragments of the proglucagon polypeptide or of GLP-1(1-37) by fragmenting the naturally-occurring amino acid sequence using,for example, a proteolytic enzyme. Further, it is possible to obtain thedesired fragments of the proglucagon peptide or of GLP-1 (1-37) throughthe use of recombinant DNA technology as disclosed by Maniatis, T., etal., Molecular Biology: A Laboratory Manual, CSH (Cold Spring Harbor,1982).

Likewise, the state of the art in molecular biology provides theordinarily skilled artisan another means by which compounds of thepresent invention can be obtained. Although it may be produced by solidphase peptide synthesis or recombinant methods, recombinant methods maybe preferable because higher yields are possible. The basic steps inrecombinant production are:

a) isolating a natural DNA sequence encoding a GLP-1 molecule orconstructing a synthetic or semi-synthetic DNA coding sequence for aGLP-1 molecule,

b) placing the coding sequence into an expression vector in a mannersuitable for expressing proteins either alone or as a fusion proteins,

c) transforming an appropriate eukaryotic or prokaryotic host cell withthe expression vector,

d) culturing the transformed host cell under conditions that will permitexpression of a GLP-1 molecule, and

e) recovering and purifying the recombinantly produced GLP-1 molecule.

As previously stated, the coding sequences may be wholly synthetic orthe result of modifications to the larger, native glucagon-encoding DNA.A DNA sequence that encodes preproglucagon is presented in Lund, et al.,Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982) and may be used asstarting material in the semisynthetic production of the compounds ofthe present invention by altering the native sequence to achieve thedesired results.

Synthetic genes, the in vitro or in vivo transcription and translationof which results in the production of a GLP-1 molecule, may beconstructed by techniques well known in the art. Owing to the naturaldegeneracy of the genetic code, the skilled artisan will recognize thata sizable yet definite number of DNA sequences may be constructed, allof which encode GLP-1 molecules.

The methodology of synthetic gene construction is well known in the art.See Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y.,Vol. 68, pgs. 109-151. DNA sequences that encode a GLP-1 molecule can bedesigned based on the amino acid sequences herein disclosed. Oncedesigned, the sequence itself may be generated using conventional DNAsynthesizing apparatus such as the Model 380A or 380B DNA synthesizers(PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City,Calif. 94404).

To effect expression of a GLP-1 molecule, one inserts the engineeredsynthetic DNA sequence in any one of many appropriate recombinant DNAexpression vectors through the use of appropriate restrictionendonucleases. See generally Maniatis et al. (1989) Molecular Cloning; ALaboratory Manual, Cold Springs Harbor Laboratory Press, N.Y., Vol. 1-3.Restriction endonuclease cleavage sites are engineered into either endof the GLP-1 molecule-encoding DNA to facilitate isolation from, andintegration into, known amplification and expression vectors. Theparticular endonucleases employed will be dictated by the restrictionendonuclease cleavage pattern of the parent expression vector to beemployed. The choice of restriction sites are chosen so as to properlyorient the coding sequence with control sequences to achieve properin-frame reading and expression of the protein of interest. The codingsequence must be positioned so as to be in proper reading frame with thepromoter and ribosome binding site of the expression vector, both ofwhich are functional in the host cell in which the protein is to beexpressed.

To achieve efficient transcription of the synthetic gene, it must beoperably associated with a promoter-operator region. Therefore, thepromoter-operator region of the synthetic gene is placed in the samesequential orientation with respect to the ATG start codon of thesynthetic gene.

A variety of expression vectors useful for transforming prokaryotic andeukaryotic cells are well known in the art. See The Promega BiologicalResearch Products Catalogue (1992) (Promega Corp., 2800 Woods HollowRoad, Madison, Wis., 53711-5399); and The Stratagene Cloning SystemsCatalogue (1992) (Stratagene Corp., 11011 North Torrey Pines Road, LaJolla, Calif., 92037). Also, U.S. Pat. No. 4,710,473 describes circularDNA plasmid transformation vectors useful for expression of exogenousgenes in E. coli at high levels. These plasmids are useful astransformation vectors in recombinant DNA procedures and

(a) confer on the plasmid the capacity for autonomous replication in ahost cell;

(b) control autonomous plasmid replication in relation to thetemperature at which host cell cultures are maintained;

(c) stabilize maintenance of the plasmid in host cell populations;

(d) direct synthesis of a protein prod. indicative of plasmidmaintenance in a host cell population;

(e) provide in series restriction endonuclease recognition sites uniqueto the plasmid; and

(f) terminate mRNA transcription.

These circular DNA plasmids are useful as vectors in recombinant DNAprocedures for securing high levels of expression of exogenous genes.

Having constructed an expression vector for a GLP-1 molecule, the nextstep is to place the vector into a suitable cell and thereby construct arecombinant host cell useful for expressing the polypeptide. Techniquesfor transforming cells with recombinant DNA vectors are well known inthe art and may be found in such general references as Maniatis, et al.supra. Host cells made be constructed from either eukaryotic orprokaryotic cells.

Prokaryotic host cells generally produce the protein at higher rates andare easier to culture. Proteins which are expressed in high-levelbacterial expression systems characteristically aggregate in granules orinclusion bodies which contain high levels of the overexpressed protein.Such protein aggregates typically must be solubilized, denatured andrefolded using techniques well known in the art. See Kreuger, et al.(1990) in Protein Folding, Gierasch and King, eds., pgs 136-142,American Association for the Advancement of Science Publication No.89-18S, Washington, D.C.; and U.S. Pat. No. 4,923,967.

Once the desired GLP-1 molecule is prepared, providing it has anisoelectric point in the range from about 6.0 to about 9.0, complexes ofthe present invention are prepared by complexing a desired GLP-1molecule with a divalent metal cation via well known methods in the art.Such metal cations include, for example, Zn⁺⁺, Mn⁺⁺, Fe⁺⁺, Co⁺⁺, Cd⁺⁺,Ni⁺⁺, and the like. Of the metal cations, Zn⁺⁺ is preferred.

Generally, a desired GLP-1 molecule, having the required isoelectricpoint, is combined with a mixture of an appropriate buffer and anappropriate form of a metal cation.

Appropriate buffers are those which will maintain the mixture at a pHrange from about 6.0 to about 9.0, but which will not interfere with thereaction. Preferred buffers include Goode's buffers, particularly HEPES,and Tris and Tris acetate.

Appropriate forms of metal cations are any form of a divalent metalcation which is available to form a complex with a GLP-1 molecule of thepresent invention. Preferably, a divalent metal cationic salt such aszinc chloride is provided in excess to provide a molar ratio of up toabout 50 molecules of a divalent metal cation for each molecule of GLP-1substrate.

The temperature employed in this step is that which is sufficient toeffect completion of the reaction. Typically, the reaction is run atambient temperature.

The product of the present reaction, a crystalline or amorphoussuspension, is isolated and purified using standard techniques.

The present invention also provides pharmaceutical compositionscomprising a compound of the present invention in combination with apharmaceutically acceptable carrier, diluent, or excipient. Suchpharmaceutical compositions are prepared in a manner well known in thepharmaceutical art, and are administered individually or in combinationwith other therapeutic agents, preferably via parenteral routes.Especially preferred routes include intramuscular and subcutaneousadministration.

Parenteral daily dosages, preferably a single, daily dose, are in therange from about 1 pg/kg to about 1,000 μg/kg of body weight, althoughlower or higher dosages may be administered. The required dosage willdepend upon the severity of the condition of the patient and upon suchcriteria as the patient's height, weight, sex, age, and medical history.

In making the compositions of the present invention, the activeingredient, which comprises at least one compound of the presentinvention, is usually mixed with an excipient or diluted by anexcipient. When an excipient is used as a diluent, it may be a solid,semi-solid, or liquid material which acts as a vehicle, carrier, ormedium for the active ingredient.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to particle size of less than about200 mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,trehalose, sorbitol, and mannitol. The compositions of the invention canbe formulated so as to provide quick, sustained or delayed release ofthe active ingredient after administration to the patient by employingprocedures well known in the art.

The compositions are preferably formulated in a unit dosage form witheach dosage normally containing from about 50 μg to about 100 mg, moreusually from about 1 mg to about 10 mg of the active ingredient. Theterm "unit dosage form" refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with a suitablepharmaceutical excipient.

For the purpose of parenteral administration, compositions containing acompound of the present invention preferably are combined with distilledwater and the pH is adjusted to about 6.0 to about 9.0.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb a compound of the presentinvention. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyesters, polyamino acids,polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose,carboxymethylcellulose, and protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release.

Another possible method to control the duration of action by controlledrelease preparations is to incorporate a compound of the presentinvention into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetatecopolymers.

Alternatively, instead of incorporating a compound into these polymericparticles, it is possible to entrap a compound of the present inventionin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules, respectively, or in colloidal drug deliverysystems, for example, liposomes, albumin microspheres, microemulsions,nanoparticles, and nanocapsules, or in macroemulsions. Such teachingsare disclosed in Remington's Pharmaceutical Sciences (1980).

The compounds of the present invention have insulinotropic activity.Thus, another aspect of the present invention provides a method forenhancing the expression of insulin comprising providing to a mammalianpancreatic B-type islet cell an effective amount of a compound of thepresent invention.

Similarly, the present invention provides a method for treating maturityonset diabetes mellitus in a mammal, preferably a human, in need of suchtreatment comprising administering an effective amount of a compound orcomposition of the present invention, to such a mammal.

The following examples are provided to further illustrate the presentinvention. It is not intended that the invention be limited in scope byreason of any of the following examples.

EXAMPLE 1

Individual aliquots of 5 different GLP-1 molecules were prepared bywell-known, solid phase peptide synthesis and were lyophilized in smallvials. Portions of O.lM HEPES(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffers at pH7.4 containing various levels of zinc chloride were added to thealiquots to obtain a protein concentration of about 0.1 mg/mL. Thesamples were mixed and stored at ambient temperature (22° C.) for about18 hours. The mixtures were then centrifuged (Fisher Model 235Cmicro-centrifuge) for 5 minutes. The clear supernatants were pipettedfrom the tubes. The protein content of the supernatants was estimated bymeasuring their absorbance at 280 nm in a spectrophotometer (Gilford260). The theoretical absorbance value for a 0.1 mg/mL solution of theGLP-1 molecules at this wavelength in the 1 cm cuvettes is 0.207. Theresults of this experiment are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Zn/GLP-1                                                                           280 nm Absorbance                                                        Molecule                 α-methly-                                                                      Gly.sup.8 -Gln.sup.21                           Molar GLP-1 Gly.sup.8 -GLP-1 Val.sup.8 -GLP-1 Ala.sup.8 -GLP-1 GLP-1                                         Ratio (7-36)NH.sub.2 (7-36)NH.sub.2                                          (7-37)OH (7-36)NH.sub.2 (7-37)OH              __________________________________________________________________________    0    0.172 0.136  0.187  0.163  0.167                                           0.3 0.099 0.079 0.191 0.134 0.113                                             0.5 0.057 0.070 0.184 0.098 0.082                                             0.7 0.035 0.058 0.180 0.079 0.069                                             1.0 0.039 0.057 0.173 0.076 0.065                                             3.0 0.048 0.044 0.110 0.055 0.055                                           __________________________________________________________________________

This example shows that only a small quantity of zinc is required tocomplex with and precipitate a significant portion of the GLP-1molecules from these dilute solutions.

EXAMPLE 2

5 mg of GLP-1 (7-36)NH₂ was completely dissolved in 2.5 mL of pH 7.4,zinc-free 0.1M HEPES buffer. An additional 2.5 mL of pH 7.4, 0.1M HEPESbuffer containing 0.6 mM zinc chloride was quickly added. Theapproximate molar ratio of zinc to GLP-1 (7-36)NH₂ in this sample is 1to 1. The solution immediately became cloudy and precipitation soonformed. The mixture was stored at ambient temperature (22° C.) for 18hours.

The precipitate became firmly attached to the bottom of the glass vial.The supernatant was completely decanted by pipette. The precipitate wasthen completely dissolved in 5.0 mL of 0.01N hydrochloric acid. Theabsorbance at 280 nm was determined for both the supernatant andredissolved precipitate solutions. The zinc levels in these solutionswere quantitated by atomic absorption spectrophotometry. The results ofthis experiment are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                                 Zinc                                                    Concentration                                                                 in Parts per                                                                 280 nm Absorbance Million                                                   ______________________________________                                        Supernatant (5 ml)                                                                            0.118        9.02                                               Redissolved Precipitate (5 ml) 1.932 13.3                                   ______________________________________                                    

This example shows that most of the GLP-1 (7-36)NH₂ precipitated fromthe solution when the zinc-containing HEPES solution was added. The 280nm absorbance value of 1.932 indicates the GLP-1 (7-36)NH₂ concentrationof the redissolved precipitate is 0.933 mg/ml, or 283 μM. The zincconcentration of this same solution, 13.3 parts per million, isequivalent to a zinc concentration of 203 μM. Therefore, the molar ratioof zinc to GLP-1 (7-36)NH₂ in the precipitate was 0.717 to 1.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 1                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 30                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <223> OTHER INFORMATION: The arginine residue at - #position 30 is                 modified so as to replace the - #terminal carboxyl group with an              amine.                                                                   - - <400> SEQUENCE: 1                                                         - - His Ala Glu Gly Thr Phe Thr Ser Asp Val Se - #r Ser Tyr Leu Glu Gly        1               5 - #                 10 - #                 15              - - Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Va - #l Lys Gly Arg                          20      - #            25      - #            30                 __________________________________________________________________________

We claim:
 1. A complex consisting of a divalent metal cation associatedwith and capable of co-precipitating with a compound of the formula:

    R.sub.1 -X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sub.2

wherein: R₁ is selected from the group consisting of L-histidine,D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, andalpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln,Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu,Gln, Ala, Thr, Ser, and Gly; R₂ is selected from the group consisting ofNH₂, and Gly-OH; providing that the compound has an isoelectric point inthe range from about 6.0 to about 9.0 and further providing that when R₁is His, X is Ala, Y is Glu, and Z is Glu, R₂ must be NH₂.
 2. A complexof claim 1 wherein said divalent metal cation is zinc.
 3. A complex ofclaim 2 wherein R₁ is chosen from the group consisting of His anddesamino-histidine.
 4. A complex of claim 2 wherein X is chosen from thegroup consisting of Ala, Gly, and Val.
 5. A complex of claim 2 wherein Yis chosen from the group consisting of Glu and Gln.
 6. A complex ofclaim 2 wherein Z is chosen from the group consisting of Glu and Gln. 7.A complex of claim 2 wherein R₁ is His, X is Val, Y is Glu, Z is Glu,and R₂ is Gly-OH.
 8. A complex of claim 2 wherein R₁ is His, X is Gly, Yis Gln, Z is Glu, and R₂ is Gly-OH.
 9. A pharmaceutical compositionwhich comprises a complex according to claim 1 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.10. A pharmaceutical composition which comprises a complex according toclaim 2 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 11. A pharmaceutical compositionwhich comprises a complex according to claim 3 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.12. A pharmaceutical composition which comprises a complex according toclaim 4 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 13. A pharmaceutical compositionwhich comprises a complex according to claim 5 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.14. A pharmaceutical composition which comprises a complex according toclaim 6 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 15. A pharmaceutical compositionwhich comprises a complex according to claim 7 in combination with oneor more pharmaceutically acceptable carriers, diluents, or excipients.16. A pharmaceutical composition which comprises a complex according toclaim 8 in combination with one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 17. A method of treating maturityonset diabetes mellitus which comprises administering to a mammal inneed of such treatment an effective amount of a complex according toclaim 1 to said mammal.
 18. A method of treating maturity onset diabetesmellitus which comprises administering to a human in need of suchtreatment an effective amount of a complex according to claim 1 to saidhuman.
 19. A method of treating maturity onset diabetes mellitus whichcomprises administering to a human in need of such treatment aneffective amount of a complex according to claim 2 to said human.
 20. Amethod of treating maturity onset diabetes mellitus which comprisesadministering to a human in need of such treatment an effective amountof a complex according to claim 3 to said human.
 21. A method oftreating maturity onset diabetes mellitus which comprises administeringto a human in need of such treatment an effective amount of a complexaccording to claim 4 to said human.
 22. A method of treating maturityonset diabetes mellitus which comprises administering to a human in needof such treatment an effective amount of a complex according to claim 5to said human.
 23. A method of treating maturity onset diabetes mellituswhich comprises administering to a human in need of such treatment aneffective amount of a complex according to claim 6 to said human.
 24. Amethod of treating maturity onset diabetes mellitus which comprisesadministering to a human in need of such treatment an effective amountof a complex according to claim 7 to said human.
 25. A method oftreating maturity onset diabetes mellitus which comprises administeringto a human in need of such treatment an effective amount of a complexaccording to claim 8 to said human.
 26. A method for enhancing theexpression of insulin comprising providing to a mammalian pancreaticB-type islet cell an effective amount of a complex of claim
 1. 27. Thepharmaceutical composition of claim 9 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 28. Thepharmaceutical composition of claim 10 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 29. Thepharmaceutical composition of claim 11 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 30. Thepharmaceutical composition of claim 12 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 31. Thepharmaceutical composition of claim 13 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 32. Thepharmaceutical composition of claim 14 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 33. Thepharmaceutical composition of claim 15 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.
 34. Thepharmaceutical composition of claim 16 wherein the complex is asubstantially insoluble crystalline or amorphous suspension whenprepared in a pharmaceutically acceptable aqueous diluent.